Antiestrogens (AEs) are the most widely used agents for the treatment of hormone-responsive breast cancer, and the AE tamoxifen has also proven to be effective in preventing breast cancer. AEs are also unique ligands useful for understanding the tissue selective actions of certain estrogens, an important issue in menopausal hormone replacement therapy, and for probing the intriguing pharmacology of the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, and their roles in breast cancer and other estrogen target cells. In this application, we are proposing to investigate two new aspects of the action of AEs. The first aspect deals with the ability of certain proteins (denoted PAAs for "Potentiators of Antiestrogen Activity") that we have identified recently, to enhance the potency of AEs as inhibitors of estrogens. The levels and activity of these small, estrogen receptor-selective proteins could account for the differential tissue selectivity of AEs and for the ability of ER-containing breast tumors to be either highly sensitive, or resistant to AE therapy. We propose to identify and characterize PAAs, by examining their roles as modulators of AE action in target cells and determinants of hormonal resistance in breast cancer, by elucidating their interaction domains with ER, their subcellular distribution, and their interaction with other ER coregulators, and by performing structural analyses on PAA-ER complexes. The second aspect also derives from our recent work in which we have found that certain antioxidant/cytoprotective genes are upregulated by AEs and inhibited by estrogens. We propose to identify and analyze the regulation of genes selectively upregulated by AEs, by searching for other genes that are AE stimulated and estrogen suppressed, by analyzing the gene regulatory regions mediating the selective activation by AEs, by determining the ERalpha and ERbeta selectivity of this regulation, and by examining whether other antioxidant genes involved in cytoprotection against reactive oxygen species are upregulated by AEs. The studies we propose should provide significant new insight into how AEs act, what cellular factors determine their effectiveness and tissue selectivity, and how their gene regulating activities contribute to their beneficial antiproliferative, tumor suppressive, and cytoprotective actions in breast cancer treatment and prevention.